Experimental Corneal Imaging and Intrastromal Tissue Ablation With Femtosecond Laser Scanning Microscopy

Abstract

Abstract: : Purpose: Amplified femtosecond (fs) laser systems emitting µJ pulse energies are novel tools for corneal refractive surgery. Studies suggest that nJ laser pulses of non – amplified fs lasers offer more precise cutting with reduced collateral tissue damage. Using a non–amplified near–infrared fs laser scanning microscope, we performed intrastromal corneal ablation and fast non – invasive multiphoton imaging at submicron resolution. Methods: A mode–locked, non–amplified Titanium:sapphire laser (Chameleon^TM, Coherent Inc., Santa Clara, CA) with a maximum output power of 1W, a tuning range of 720–930 nm, and a repetition rate of <140 fs was coupled to a laser scanning microscope (LSM510^TM, Zeiss, Jena, Germany). The system was used to image and cut human pathologic corneas obtained from keratoplasty. The laser beam was focused through a 40x oil–immersion objective (Zeiss Neofluar^TM, Zeiss, Jena, Germany) with a numeric aperture of 1.3. Results: Different wavelengths (720 to 880 nm) and laser pulse energies were tested with respect to precision and collateral damage of intrastromal tissue ablation. The lesion width was below 5 µm. The laser scanning microscope allowed to investigate different corneal pathologies with multiphoton imaging. A spectral analysis was performed. Native autofluorescence, second harmonic generation, and luminescence along laser tissue cuts were correlated with transmission electron microscopy images. Human corneal pathology included Fuchs' endothelial dystrophy, keratoconus, herpes keratitis, and corneal stromal scars. Conclusions: Laser scanning microscopy with non – amplified fs laser pulses allows precise tissue removal with limited collateral damage. In addition, functional images allow verification of tissue ablation within seconds. Pathologic corneal tissues show specific morphologic and spectral features. Future projects will study corneal wound healing in an animal model and refine the diagnostic potential of corneal laser scanning microscopy in vivo.

Keywords: laser • microscopy: light/fluorescence/immunohistochemistry • cornea: clinical science